Acute leukemia and lymphoblastic lymphona-specific cd43 epitope and use thereof

ABSTRACT

The present invention relates to a CD43 epitope expressed on human acute leukemia and lymphoblastic lymphoma cells and its use. More particularly, the present invention relates to a CD43 epitope expressed on human acute leukemia, lymphoblastic lymphoma cells, but not on mature hematopoietic cells, hematopoietic stem cells and non-hematopoietic cells, and to its diagnostic and therapeutic application on acute leukemia and lymphoblastic lymphoma.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to and the benefit of U.S. provisionalapplication No. 60/679,910 filed in the United State of Patent &Trademark Office on May 11, 2005 and of Korean Patent Application. No.2005-0077906 filed in the Korean Intellectual Property Office on Aug.24, 2005, the entire content of which are incorporated hereinto byreference.

FIELD OF THE INVENTION

The present invention relates to a CD43 epitope expressed on human acuteleukemia and lymphoblastic lymphoma cells and its use. Moreparticularly, the present invention relates to a CD43 epitope exposed onhuman acute leukemia and lymphoblastic lymphoma cells, but not on maturehematopoietic cells and hematopoietic stem cells, and to its diagnosticand therapeutic application on acute leukemia and lymphoblasticlymphoma.

BACKGROUND OF THE INVENTION

The CD43 molecule, also called sialophorin or leukosialin, is acell-surface molecules expressed on most of hematopoietic cells excepterythrocytes. Human CD43 has a mucin-like extracellular domain of 235amino acids (aa), a transmembrane domain of 23 as and a 123 asintracytoplasmic domain, all encoded by one exon (Pallant et al., ProcNatl Acad Sci USA. 1989, 86:1328-32; Shelley et al., Proc Natl Acad SciUSA 1989, 86:2819-23). The extracellular domain of human CD43 is richthe amino acids serine (46 residues) and threonine (47 residues), mostof which carries about 80 0-linked carbohydrate chains. In addition,CD43 carries 1 N-linked carbohydrate chain. The structure of theseO-glycans varies from one cell type to another (Carlsson et al., J BiolChem. 1986, 261:12787-95).

The CD43 gene consists of two exons, separated by a 378 by intron,whereby the entire translation product is encode by the second exon(Shelley et al., Biochem J. 1990, 270:569-76). CD43 has been believed tobe a specific leukocyte-type marker restricted to most of leukocytes,platelets and hematopoietic stem cells, except for erythrocytes(Remold-O'Donnell et al., Blood. 1987, 70:104-9; Fukuda, Glycobiology.1991, 1:347-56). However, the expression of CD43 in human tumor cells ofnon-hematopoietic origin, such as a human uterine cervix cancer cellline (CaSKI), a human lung cancer cell line (A549), a human breastadenocarcinoma cell line (MCF7), a human fibrosarcoma cell line (HT1080), and human colonic adenocarcinoma cell lines (COLO 205, HT 29,Caco-2, DLD-1 and SW480), has been demonstrated (Fernandez-Rodriguez etal., Tumour Biol. 2002, 23:193-201). CD43 is also expressed in humancolon cancer tissue (Sikut et al, Int J. Cancer. 1999, 82:52-8; Jung etal, Korean J Pathol. 38:8-14).

Biosynthetic studies show that the CD43 precursor, with a predicted sizeof around 40 kDa (including one N-glycan), migrates with an apparentmolecular mass of 54 kDa upon electrophoresis. This precursor issubsequently converted to a mature glycosylated molecule with sized from115 kDa to up to more than 200 kDa due to variable glycosylation.Thymocytes, CD4+T lymphocytes and monocytes express more of a 115 kDaisoform, whereas a 130 kDa form is found mostly on activated CD4+Tcells, CD8′ resting and activated T cells, neutrophils, platelets and Bcells (Rosenstein et al., Immunol Res. 1999, 20:89-99). It seems thatmore than one isoform can be co-expressed on the surface of the same cc11. A tightly regulated post-translational 0 glycosylation patternresults in these characteristic molecular weight isoforms that aredifferentially expressed in different cell types. Especially, expressionof core 2β-1, 6-N-acetylglucosaminyltransferase (C2GnT) results inexpression of the 130 kDa CD43 isoform in thymocytes and T cells (Pilleret al., J Biol. Chem. 1988, 263:15146-50).

Until recently, more than 17 anti-human CD43 antibodies had beenreported. Most of these antibodies react with carbohydrate epitopes onthe extracellular domain and all known anti-CD43 antibodies detect theCD43 protein expressed on mature hematopoietic cells (Table 1). Thus,they are not efficient at detecting or eradicating leukemic or lymphomacells.

TABLE 1 Anti-CD4 antibody CD43-positve cells CD43-negative cells Epitope*Reference T305 Activated CD4⁺ T cells, Granulocytes, Core-2 1, 2 CD8⁺ Tcells, erythrocytes, platelets thymocytes, myeloid carbohydrateprecursors in bone marrow L10 T cells, thymocytes, B Erythrocytes 1-78Sialidase- 3 cell lines, monocytes, resistant neutrophils, platelets L2T cells, thymocytes, B Erythrocytes 1-78 cell lines, monocytes,neutrophils, platelets 84-3C1 Bone marrow cells, Platelets, erythrocytesSialidase-sensitive 4 thymocytes, T cells, carbohydrate monocytes,granulocytes MEM-59 Most of leukocytes, Neuraminidase- 5, 6 CD34⁺ bonemarrow sensitive cells carbohydrate MT-1, L60 T cells, monocytes B cellsNeuraminidase- 7 DFT1 T cells, monocytes B cells sensitive carbohydrate1G10 T cells, NK cells, B cells, Erythrocytes 8 granulocytes CBF.78 Tcells, subsets of Neuraminidase- monocytes and resistant granulocytesRDF/AD-9, T cells, monocytes, B cells Neuraminidase- 9 granulocytessensitive carbohydrate 161-46 T cells, monocytes, B cells Neuraminidase-granulocytes resistant 4D2 COLO 205, K562, intracellular 10  Jurkat(337-343) 4D1 Activated CD4⁺ T cells, B cells Core-2 Mukasa et monocytescarbohydrate al., 1999 *1. Fox et al., J Immunol. 1983, 131: 762-7; 2.Saitoh et al., Blood. 1991, 77: 1491-9; 3. Remold-O'Donnell et al.,Blood. 1987, 70: 104-9; 4. Borche et al., Eur Jlmmunol. 1987, 17:1523-6; 5. Stefanova et al., Folia Biol (Praha). 1988, 34: 255-65; 6.Alvarado et al., Eur J Immunol. 1995, 25: 1051-5; 7. Stross et al, JClin Pathol. 1989, 42: 953-61; 8. Horejsi et al., 1997, In Kishimoto T,et at, Leucocyte Typing, Vol. VI: White Cell Differentiation Antigens494. Garland, New York and London; 9. Tkaczuk et al., Tissue Antigens.1999, 54:1-15; 10. Sikut et al., Int J Cancer. 1999, 82: 52-8; 11.Mukasa et al, Int Immunol. 1999, 11: 259-68.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an acute leukemia,chronic leukemia with blast crisis and lymphoblastic lymphoma-associatedCD43 epitope where the epitope is exposed on human acute leukemia andlymphoblastic lymphoma cells, but not on mature hematopoietic cells andhematopoietic stem cells. Thus, the present invention provides anisolated polypeptide for epitope of CD43 comprising an amino acidsequence of SEQ ID NO: 1.

Another object of the present invention is to provide material thatrecognizes the CD43 epitope. Preferably, the present invention providesan antibody or its fragment which specifically binds to an epitope ofCD43 comprising an amino acid sequence of SEQ ID NO: 1.

A third object of the present invention is to provide a method forproducing the materials that recognize the CD43 epitope.

A fourth object of the present invention is to provide material for thediagnosis of acute leukemia, chronic leukemia with blast crisis, andlymphoblastic lymphoma.

A fifth object of the present invention is to provide a method ofdiagnosing acute leukemia, chronic leukemia with blast crisis, andlymphoblastic lymphoma. Thus, the present invention provides a method ofdiagnosing acute leukemia comprising leukemia cells in a biologicalsample with anti-CD43 epitope antibody, and detecting the positivereaction to the anti-CD43 epitope antibody. The present inventionprovides a method of diagnosing chronic leukemia with blast crisiscomprising incubating leukemia cells in a biological sample withanti-CD43 epitope antibody, and detecting the positive reaction to theanti-CD43 epitope antibody. The present invention provides a method ofdiagnosing lymphoblastic lymphoma comprising incubating lymphoma cellsin a biological sample with anti-CD43 epitope antibody according toclaim 6, and detecting the positive reaction to the anti-CD43 epitopeantibody.

A sixth object of the present invention is to provide a pharmaceuticalcomposition which can kill tumor cells of acute leukemia, chronicleukemia with blast crisis and lymphoblastic lymphoma.

A seventh object of the present invention is to provide a method fordiagnosis by use of the diagnostic material of the present invention.

In addition, the present invention is to provide a method for treatmentby use of the therapeutic material of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention, and many of the attendantadvantages thereof, will be readily apparent as the same becomes betterunderstood by reference to the following detailed description whenconsidered in conjunction with the accompanying drawing, wherein:

FIG. 1 is a photograph of immunohistochemical staining of thymus bysupernatant of hybridoma clone producing EB-1 monoclonal antibodyspecific against the CD43 epitope.

FIG. 2 is a photograph of the reactivity of EB-1 monoclonal antibody onthe surface of human thymocytes using triple color flow cytometry.

FIG. 3 is a photograph of the reactivity of EB-1 monoclonal antibody onthe surface of human peripheral blood leukocytes and hematopoietic stemcells of cord blood using triple color flow cytometry.

FIG. 4 is a photograph of the reactivity of EB-1 monoclonal antibody onthe surface of human bone marrow cells using triple color flowcytometry.

FIG. 5 is a photograph of the reactivity of BB-1 monoclonal antibody onthe surface of human CD43-transfected 29T cell line using single colorflow cytometry.

FIG. 6 is a photograph of the reactivity of EB-1 monoclonal antibody onthe surface of human Molt-4 cell line with or without sialidasetreatment using single color flow cytometry.

FIG. 7 is a photograph of SDA-PAGE analysis of human thymocyte andMolt-4 cell line lysate with or without sialidase treatment, followed byimmunoblotting with EB-1 monoclonal antibody.

FIG. 8 is a schematic diagram of 11 types of CD43 deletion mutants.

FIG. 9 is a photograph of SDA-PAGE analysis of 11 types of CD43 deletionmutants, followed by immunoblotting with EB-1 monoclonal antibody andanti-GST polyclonal antibody.

FIG. 10 is a photograph of the reactivity of EB-1 monoclonal antibody onthe surface of human leukemic cells using single color flow cytometry.

FIG. 11 is a photograph of immunohistochemical staining of lymphoblasticlymphoma tissue by EB-1 monoclonal antibody.

FIG. 12 is a photograph of the reactivity of EB-1 and anti-human MHCclass I monoclonal antibodies using dual color flow cytometry on thesurface of leukemic cells in the peripheral blood of RAG-1-deficientmice in which CCRF-CEM cells were intravenously injected and treatedwith EB-1 or control antibody.

FIG. 13 is a photograph of the reactivity of two additional anti-CD43antibodies (EB-2 and EB-3), which were produced by immunization of micewith CD43 epitope, on the surface of CD43-transfected ETA cell line,human thymocytes, human peripheral blood leukocytes, and human leukemiasample using single color flow cytometry.

FIG. 14 is a photograph of SDA-PAGE analysis of 5 types of CD43 deletionmutants, followed by immunoblotting with EB-2 and EB-3 monoclonalantibodies.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention is to provide a CD43 epitope exposed on the tumorcells of acute leukemia, chronic leukemia with blast crisis andlymphoblastic lymphoma, but not on mature hematopoietic cells andhematopoietic stem cells. The CD43 of the present invention is apreferably human CD43, which sequence had been submitted to the NCBIGenBank under accession No. M61827. The CD43 epitope is any polypeptidethat includes amino acid sequence in SEQ ID NO: 1 (hereinafter, EP6),and more preferably includes amino acid sequence in SEQ ID NO: 2(hereinafter, EP9).

EP6: Pro Leu Tip Thr Ser Ile (SEQ ID NO: 1)

EP9: Glu Gly Ser Pro Leu Tip Thr Ser Ile (SEQ ID NO: 2).

In a preferred embodiment, the present invention provides a polypeptideof less than 200 amino acids in length, more preferably less than 100amino acids in length, more preferably less than 50 amino acids inlength, comprising an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

The present invention also provides a polypeptide consisting essentiallyof an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. The presentinvention also provides a polypeptide consisting of an amino acidsequence of SEQ ID NO:1 or SEQ ID NO:2.

The CD43 epitope (EP6 and EP9) could be purified from human tissues,chemically synthesized, or produced by biotechnology.

Also, the present invention is to provide material recognizing the CD43epitope of the present invention. The material is selected from thegroup consisting of antibody, its fragment, and ligands, and theantibody is preferably selected from the group consisting of monoclonaland polyclonal antibody, and more preferably it is originated from humanand animal.

The antibodies comprise parts of antibody including antigen recognitionregion (_(VH) and V_(L)), so has the capacity to recognize antigenpreferentially. In addition, it also includes antibody fragment, such asF(ab′)2, Fab and Fv. Preferentially the antibody fragment (Fv) comprisesa single chain polypeptide fragment of antibody, so called single chainFv, which is prepared by inserting a linking peptide between twopolypeptides, V_(H) and V_(L) to increase heat stability.

The present invention further provides chimeric antibodies recognizingthe CD43 epitope of the present invention. As used herein, the term“chimeric antibody” refers to an antibody in which the variable regionsof antibodies derived from one species are combined with the constantregions of antibodies derived from a different species or alternativelyrefers to CDR grafted antibodies. Chimeric antibodies are constructed byrecombinant DNA technology, and are described in Shaw, et al., J.Immun., 138:4534 (1987), Sun, L K., et al., Proc. Natl. Acad. Sci. USA,84:214-218 (1987), for example.

Other known techniques can be used to generate CDR grafted and chimericantibodies. “CDR” or “complementarity determining region” or“hypervariable region” is defined as the amino acid sequences on thelight and heavy chains of an antibody which form the three-dimensionalloop structure that contributes to the formation of the antigen bindingsite.

As used herein, the term “CDR grafted” antibody refers to an antibodyhaving an amino acid sequence in which at least parts of one or more CDRsequences in the light and/or variable domain have been replaced byanalogous parts of CDR sequences from an antibody having a differentbinding specificity for a given antigen or receptor.

The terms “light chain variable region” and “heavy chain variableregion” refer to the regions or domains at the N-terminal portion of thelight and heavy chains respectively which have a varied primary aminoacid sequence for each antibody. The variable region of the antibodyconsists of the amino terminal domain of the light and heavy chains asthey fold together to form a three-dimensional binding site for anantibody.

The analogous CDR sequences are said to be “grafted” onto the substrateor recipient antibody. The “donor” antibody is the antibody providingthe CDR sequence, and the antibody receiving the substituted sequencesis the “substrate” antibody. One of skill in the art can readily producethese CDR grafted antibodies using the teachings provided herein incombination with methods well known in the art (see Borrebaeck, C. A.,Antibody Engineering: A Practical Guide, W.H. Freeman and Company, NewYork, 1992, incorporated by reference).

This invention further provides humanized antibodies recognizing theCD43 epitope of the present invention. As used herein, the term“humanized antibody” refers to forms of antibodies that containsequences from non-human (e.g., murine) antibodies as well as humanantibodies. Such antibodies are chimeric antibodies which containminimal sequence derived from non-human immunoglobulin. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe hypervariable loops correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin sequence. The humanized antibody optionallyalso will comprise at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. See e.g., CabillyU.S. Pat. No. 4,816,567; Queen et al. (1989) Proc. Nat'l Acad. Sci. USA86:10029-10033; and ANTIBODY ENGINEERING: A PRACTICAL APPROACH (OxfordUniversity Press 1996).

In one preferred embodiment of the present invention, the antibody bindsto a peptide consisting of the amino acid sequence shown in SEQ ID NO:1.

The antibody or its fragment may further comprise labeling materialwhich is selected from the group of radioisotopes, toxins, fluorescentmaterials and staining materials. The examples of the fluorescentmaterials are fluorescein-5-isothiocyanate (FITC), phycoerythrin (PE),allophycocyanin (APC), and biotin, but does not limited thereto.

The antibody or its fragment may further comprise a toxic materialselected from the group consisting of radioisotopes, toxic chemicals,toxic proteins and anti-tumor agents. The antibody or its fragment iscombined with the toxic proteins to produce a fusion protein. Theexamples of the toxic proteins include ricin, saporion, gelonin,momordin, diphtheria toxoid and pseudomonas toxin, but does not limitedthereto.

The present invention also provides a method for producing the material,and it provides cells producing the antibody. The method of producingantibody or its fragment includes the steps of (a) immunizing an animalwith polypeptide, protein or protein fragments containing or cellsexpressing the CD43 epitope, (b) extracting splenocytes from theimmunized animal, (c) fusing the splenocytes with myeloma cells, and (d)screening hybridoma cells producing antibodies against the CD43 epitope.The material can be obtained by in vitro culture or injection into theanimals of cells producing the materials. The material can be obtainedfrom the ascites of animals in which the cells producing the materialsare intraperitoneally injected. The materials can be purified from theculture supernatant or ascites by ion exchange chromatography oraffinity column chromatography.

In an embodiment, the present invention relates to a pharmaceuticalcomposition for treating the acute leukemia, the chronic leukemia withblast crisis, lymphoblastic lymphoma comprising an anti-CD43 epitopeantibody or its fragment, and a pharmaceutically acceptable carrier.

Administration of the antibody or its fragment according to theinvention can be carried out using any of the accepted modes ofadministration of pharmaceutical compositions. Thus, administration canbe, for example, The antigen recognition material can be administeredorally or 10 more preferably parenterally into the patients, in the formof solid, semi-solid, lyophilized powder, or liquid dosage forms, suchas, for example, tablets, suppositories, pills, soft elastic and hardgelatin capsules, powders, solutions, suspensions, or aerosols, or thelike, preferably in unit dosage forms suitable for simple administrationof precise dosages. The pharmaceutical compositions will generallyinclude a conventional pharmaceutical carrier or excipient and theantibody of the invention as the/an active agent, and, in addition, mayinclude other medicinal agents, pharmaceutical agents, carriers,adjuvants, diluents, vehicles, or combinations thereof. Suchpharmaceutically acceptable excipients, carriers, or additives as wellas methods of making pharmaceutical compositions for various modes oradministration are well-known to those of skill in the art. The suitabledosage of the active component can be selected depending on thecondition of the subject, for example 3 mg to 6,000 mg per 1 day.

In an embodiment, the present invention relates to a method of treatingthe acute leukemia, the chronic leukemia with blast crisis,lymphoblastic lymphoma using the antibody or its fragment, which isselected from the group consisting of antibody, antibody fragment,single chain polypeptide antibody fragment and ligand. The antibody orthe antibody fragment is preferably selected from the group consistingof monoclonal and polyclonal antibodies, and more preferably it isoriginated from human and animal. Preferably the antibody or itsfragment further includes toxic material which is selected from thegroup consisting of radioisotopes, toxic chemicals, toxic proteins andanti-tumor agents.

Further, the present invention provides a method for the diagnosis ofacute leukemia, chronic leukemia with blast crisis and lymphoblasticlymphoma using the antibody or its fragment. The method includes thesteps of (a) incubating the antibody or its fragment with a cell in abiological sample, and (b) detecting the sample showing positivereaction against the antibody. The tumor is preferably acute leukemia,chronic leukemia with blast crisis or lymphoblastic lymphoma.

In addition, the present invention provides a diagnostic kit for acuteleukemia, chronic leukemia with blast crisis and lymphoblastic lymphomausing the material. The diagnostic kit could include the method for thedetection of antigen-antibody reaction in addition to the material. Thedetection method is preferably selected from the group consisting offlow cytometry, immunohistochemistry, enzyme-linked immunosorbent assay(ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescenceimmunoassay (FIA) and luminescence immunoassay (LIA). The detectionmethod include the labeling materials, which could be selected from thegroup of enzymes (such as horse radish peroxidase (HRP)), fluorescentmaterials (such as FITC), luminescent materials (such as luminol,isoluminol and lucigenin), and isotopes (such as ¹²⁵I, ³H, ¹⁴C and¹³¹I), but is not to be considered limiting thereof in any way. Thereactivity of the antigen recognition material could be confirmed usingdevice detecting the enzyme reaction, fluorescence, luminescence, orradiation. The diagnosis kit can be made in flow cytometry kit,immunohistochemistry kit, ELISA kit or strip kit including the antibodyor its fragment.

The present invention is further explained in more detail with referenceto the following examples. These examples, however, should not beinterpreted as limiting the scope of the present invention in anymanner.

Example 1

In order to discover a specific cell surface protein on thymocytes,human thymocytes were administrated into Balb/c mice to produceantibodies to human thymocytes by the following examples.

10⁷ of human thymocytes were intraperitoneally administrated andimmunized into Balb/c mice at two weeks interval for six weeks. Thespleen of Balb/c mice was removed 3 days after last administration toprepare the spleen cell suspension.

Monoclonal antibodies were produced by fusing the spleen cells of Balb/cimmunized with human thymocytes with SP2/0-Ag14 mouse myeloma cellsresistant to 9-azaguanine. Cell fusion method followed Koeler andMilstein method (Koeler & Milstein Nature, 1975, 256, 495-497). 10⁸spleen cells were fused with 10⁷ myeloma cells using 50% polyethyleneglycol 4000. The cells were washed and resuspended in Dulbeco's modifiedEagle's medium (DMEM) containing 20% bovine serum albumin, 100 μMhypoxanthine, 0.44 μM aminopterin and 16 μM thymidine (HAT media). Thecells were introduced to four 96-well plates and cultured at 37

, and 5% CO₂ incubator. When colonies were formed after two weeks, thesupernatant was prepared and the reactivity of antibody was observedusing immunohistochemistry and flow cytometry.

The well containing more than 10⁵ cells per well was regarded aspositive group. Cells were taken from the well containing highlyreactive antibody, and subcloned 0.5 cell per well by limiting dilutionassay to produce stable hybridoma clone with high reactivity ofantibody. This hybridoma clone secretes antibody into the media and thesupernatant was stored for the next steps.

Example 2

In order to discover a clone which secrets antibody recognizing thespecific cell surface antigen on thymocytes among the hybridoma clonesproduced by Example 1, the immunoperoxidase staining was carried out onthe slide of 4 μm thick of fresh tissue and formalin-fixedparaffin-embedded tissue using the supernatant of the hybridoma cloneproduced by Example 1 according to avidin-biotin complex (ABC) stainingmethod by binding avidin with biotin. The supernatant of monoclonal cellwas used as primary antibody. The paraffin embedded tissue was treatedwith normal mouse serum and allowed to stand for 1 hour to preventnonspecific background staining after removal of paraffin. After addinga primary antibody, they were allowed to stand overnight and washedthree times with phosphate buffered saline (PBS). Biotinylated goat antimouse immunoglobulin used as a secondary antibody was added. It wasallowed to stand at room temperature for 1 hour, washed three times withPBS. Then streptavidine and horse-radish peroxidase conjugate was added.3,3′-diaminobezidine tetrahydrochloride (DAB) and H₂O₂ solutionmanufactured by DAKO was added to stain cells, treated for 20 minutesand washed three times with PBS. It was observed under light microscopyafter covering with cover glass.

Hybridoma clone lines producing antibody specific for human thymocyteswere selected. One of clones whose antibody recognized thymocytes wasnamed as EB-1. FIG. 1 is a photograph of immunohistochemical stainingthymus with the supernatant of hybridoma clone producing EB-1 antibody.As shown in FIG. 1, most of thymocytes is positively stained. Cellsurface of thymocytes were strongly stained so the antigen recognized byEB-1 antibody is cell surface antigen.

Example 3

To evaluate the reactivity of EB-1 antibody according the developmentalstages of thymocytes, flow cytometry was carried out. Human thymuses,which were removed from patient during cardiac surgery, were finelyteased, and single cell suspension was prepared. 1×10⁶ cells weresuspended in 100 pa PBS, and distributed into test tubes. 100 μl EB-1culture supernatant was added and stirred. The solution was reacted at4° C. for 30 minutes, centrifuged at 1,500 rpm for 5 minutes, and thepellet was washed twice with PBS to remove the unreacted antibody. Thepellet was suspended in 50 μl solution containing diluted secondaryantibody (FITC-conjugated goat anti-30 mouse Ig manufactured by Zymed,reacted at 4

for 30 minutes in a dark room. After washing twice, the pellet wassuspended in 50 μl solution containing phycoerythrin (PE)-conjugatedanti-CD8 antibody and allophycocyanin (APC)-conjugated anti-CD4antibody, reacted at 4

for 30 minutes in a dark room, and then washed twice. Finally 200 of PBSwas added to the cell pellet after centrifugation. Thymocytes areclassified into four subsets according to the expression pattern of CD4and CD8 (that is, CD4 CD8 double negative thymocytes, CD4⁺CD8⁺ doublepositive thymocyte, and CD4⁺CD8 or CD4CD8⁺ single positive thymocytes)and the ratio of EB-1-positive cells and the intensity and EB-1-stainingwere analyzed by flow cytometry. FIG. 2 shows the results of three colorflow cytometric analyzing showing the reactivity of BB-1 against allsubsets of thymocytes, particularly the highest staining intensity ondouble positive thymocytes. In FIG. 2, the solid line represents thestaining by EB-1 antibody, and the negative staining profile by anirrelevant antibody is shown as filled histogram.

Example 4

In order to obtain high concentration of antibody secreted byEB-1-screting hybridoma clone, ascites were prepared. Three weeks after0.5 ml of pristine was administrated intraperitoneally into Balb/cmouse, 10⁷ of EB-1 hybridoma clone cultured in DMEM containing 10%bovine serum. After 2 to 3 weeks, ascites were collected. Then theconcentration of antibody was 5 to 10 mg/ml. Only immunoglobulinsresponding to human thymocytes were purified because there are manycontaminating proteins such as albumin in ascites. To purify antibodyfrom ascites containing high amount of antibodies obtained from Balb/cmouse into which EB-1 monoclonal hybridoma cells were intraperitoneallyadministered, Q-Sepharose chromatography and hydroxyapatite (Bio-gel HTPGel manufactured by Pharmacia) chromatography were performed.

3.14 g of ammonium sulfate per 10 ml of ascites was slowly added on ice(precipitated with 50% of (NH4)₂ SO₄). The mixture was centrifuged at15,000 rpm for 30 minutes, resuspended in deionized water and dialyzedin 1 liter of buffer solution (20 mM phosphate, pH 7.4). The solutionwas passed and absorbed in Q-Sepharose column previously equilibratedwith buffer solution (20 mM phosphate, pH 7.4), and then the buffersolution was again passed through the column to remove free proteins inthe column, after which the protein absorbed in column was eluted withlinear gradient 0 M to 0.8 M of NaCl using buffer solution I (20 mMphosphate, pH 7.4) and buffer solution II (20 mM phosphate and 8.5 MNaCl, pH 7.4). Each fraction was electrophoresized in SDA-PAGE and thefractions containing EB-1 antibody were collected.

The fractions were then dialyzed in buffer solution (20 mM phosphate, pH6.8), and put through hydroxyapatite column previously equilibrated withbuffer solution (20 mM phosphate, pH 6.8). The fraction in buffersolution (20 mM phosphate, pH 6.8) passed through the column to removefree proteins and was eluted with a linear gradient 0 to 0.3 M ofphosphate using buffer solution III (20 mM phosphate, pH 6.8) and buffersolution (300 mM phosphate, pH 6.8). Each fraction was electrophoresizedin SDS-PAGE and the fractions containing more than 95% of EB-1 antibodywere collected. EB-1 antibody collected was dialyzed in appropriatebuffer solution and stored. 5 to 10 mg BB-1 antibody was prepared from 1ml of ascites by repeated experiments.

Example 5

The present example was carried out according to flow cytometricanalysis of Example 3 to investigate whether EB-1 antibody is reactiveagainst normal leukocytes except thymus using EB-1 antibody purifiedfrom Example 4 as primary antibody. For this analysis, the purified EB-1antibody was directly combined with FITC or PE, in which case it was notnecessary to use the secondary antibody for fluorescence, orbiotin-conjugated EB-1 antibody was used combined withfluorescent-conjugated streptavidine.

Table 2 below shows the reactivity of EB-1 antibody on the cell surfaceof normal peripheral blood leukocytes, activated peripheral bloodmononuclear cells cultured in the medium containing 10 μg/ml ofPhytohemagglutinine (PHA) or 5 μg/ml of anti-CD3 antibody, normal spleencell, normal thymocytes, and CD34⁺AC 133⁺ hematopoietic stem cells incord blood. All of them were negative to EB-1 antibody, except forthymocytes. The representative results of flow cytometric analysis ofnormal leukocytes are shown in FIG. 3, in which the solid linerepresents the staining by EB-1 antibody, and the negative stainingprofile by an irrelevant antibody is shown as filled histogram.

TABLE 2 Cells EB-1-Positivity Peripheral blood cells Erythrocytes −*Lymphocytes − Monocytes − Granulocytes − Platelets − Activatedperipheral blood PHA (10 μg/ml) − Anti-CD3 antibody (5 μg/ml) −Splenocytes − Thymocytes ++* CD34⁺AC 133⁺ hematopoietic stem − cells incord blood *Negative staining **Positive staining in more than 90% ofcells PHA, Phytohemagglutinine

Example 6

The present example was carried out according to immunohistochemicalassay of Example 2 to confirm whether BB-1 antibody is reactive againstnormal tissue except thymus using EB-1 antibody purified from Example 4as primary antibody. Table 3 below is the reactivity of EB-1 antibody ineach of the various tissues. Except for thymocytes, all other tissuesincluding peripheral lymphoid tissue, cerebellum, pancreas, ovary andtestis, skin, lung, adrenal, and kidney were negative for staining.

TABLE 3 No. of EB-1 Organ No. of cases positive Lymphoid System Lymphnode 18 0 Tonsil 3 0 Thymus 6 6 Spleen 4 0 Nervous System Cerebrum 4 0Cerebellum 4 0 Digestive System Esophagus 3 0 Stomach 10 0 Smallintestine 2 0 Large intestine 2 0 Liver 7 0 Pancreas 4 0 Vermiformappendix 4 0 Reproductive System Testis 2 0 Ovary 8 0 Uterus 4 0 TheOthers Lung 8 0 Kidney 9 0 Adrenal gland 5 0 Skin 4 0

Example 7

The present example was carried out according to flow cytometricanalysis of Example 3 to whether EB-1 antibody is reactive againstnormal bone marrow cells using EB-1 antibody purified from Example 4 asprimary antibody. For this analysis, biotin-conjugated EB-1 antibody andAPC-conjugated streptavidine was used in combination withFITC-conjugated anti-CD10, anti-CD33, anti-CD38, anti-CD71, oranti-AC133, and PE-conjugated-anti-CD34. FIG. 4 is the result of threecolor flow cytometric analysis, in which CD34⁺ cells were gated.Pluripotent stem cells were defined as CD34⁺AC133⁺ (FIG. 4A) orCD34⁺CD38^(−/dim) (FIG. 4B) cells in bone marrow or cord blood and theydid not show reactivity against EB-1 antibody at all. In addition,CD34⁺CD33⁺ cells (FIG. 4D) that contain virtually all the colony-formingcells such as progenitor cells capable of forming granulocytes,erythrocytes, monocytes, megakaryocytes (CFU-GEMM), CFU-GM, andburst-forming unit erythrocytes, and CD34⁺CD71^(bright)erythroid-committed progenitor cells (FIG. 4E) were also not stained byEB-1 antibody. In contrast, positive staining of EB-1 antibody wasobserved on the majority of lymphoid-committed CD34⁺CD10⁺ precursors(FIG. 4C).

In summary of the results, EB-1-reactivity was restricted to thymocytesand some hematopoietic precursors in bone marrow, but not found in anyanother hematopoietic cells including mature peripheral blood cells andhematopoietic stem cells and non-hematopoietic tissues.

Example 8

In order to clarify the antigen recognized by EB-1 antibody, a cDNAlibrary of human thymocytes was prepared using poly(A)+ RNA and thepcDNA3 expression vector manufactured by Invitrogen. This librarycontained 3.5×106 independent colonies. The strain of bacterial hostused was Escherichia coli MC1061/P3. Plasmid pMIK/D3T 31 was constructedby inserting XhoI fragment of Gb3 synthase cDNA clone, pD3T 31(Haraguchi et al., Proc. Natl. Acad. Sci. U.S.A. 1994, 91: 10455-9),into pMIK/Neo expression vector.

Plasmids of the cDNA library were once amplified and transfected into293T cells together with plasmid pMIK/D3T-31 using DEAE-dextran asdescribed previously (Davis et al., Methods in Molecular Biology,Elsevier Science, New York 1986:285-289). Subconfluent 293T cells,1.5×10⁶ in 10-cm dishes were co-transfected with 8 μg each of cDNAlibrary plasmid and pMIK/D3T-31. After 60 h, the transfected cells weredetached from plates and incubated with EB-1 antibody at a 1:200dilution on ice for 45 minutes. Cells were plated on dishes coated withgoat anti mouse IgM as described previously (Wysocki et al., Proc. Natl.Acad. Sci. U.S.A. 1978, 75:2844-8). Plasmid DNA was rescued from thepanned cells and transformed into 293T. Expanded plasmid DNA wastransfected again, and the same procedure was repeated four times more.Thereafter 96 pools containing 30 colonies each were prepared andscreened by EB-1 reactivity. Finally, 17 clones from two positive poolswere screened, and three single colonies that directed the EB-1reactivity on 293T were isolated using microscale DEAE-dextrantransfection and immunofluorescence assay.

Isolated cDNA plasmid was digested by XhoI and HindIII and cloned intophagemid BlueScript (pBSK) KS(+) vector. Deletion mutants of this clonewere prepared with a Kilo-Sequence deletion kit. Dideoxynucleotidetermination sequencing was performed by either T3/T7 dye primers or fouradditional custom dideoxy terminators with the PRISM dye terminatorcycle sequencing kit and model 377 DNA sequencer manufactured by AppliedBiosystems. Blast search using the DNA sequence identified through thisprocedure revealed that the antigen recognized by EB-1 antibody is humanCD43.

To confirm that the EB-1 antibody recognized human CD43 antigens, humanCD43 transfected 293T cells were stained with EB-1 antibody and a wellknown anti-CD43 antibody, DFT-1. As shown in FIG. 5, EB-1 antibody wasnot reactive against wild-type 293T cells, whereas CD43 transfected 293Tcells were stained by both EB-1 and DFT 1 antibodies. Thus, EB-1 is amonoclonal antibody against human CD43.

As CD43 is heavily glycosylated protein, we investigated whethersialidase treatment of CD43 molecules modify the immunoreactivity ofEB-1 antibody against CD43 antigen. FIG. 6 shows a flow cytometricanalysis of Molt-4 cells with or without sialidase treatment. The dottedline represents the negative staining profile by an irrelevant antibody,and thin and thick solid lines represent the immunoreactivity of EB-1antibody on the sialidase-untreated and treated Molt-4 cells,respectively. The immunoreactivity of BB-1 antibody on Molt-4 cells wasnot affected by sialidase treatment.

To confirm these results, SDS-PAGE and Western blotting was carried out.1×10 of human thymocytes or Molt-4 tumor cells with or without sialidasetreatment were suspended in 1 ml of lysis buffer solution (50 mMTris-HCl, pH 74, 150 mM NaCl, 0.5% w/v Nonidet P-40 and 1 mMphenylmethylsulfonyl fluoride (PMSF)), shaken at 4° C. for 30 minutes,and centrifuged at 13,000 g for 15 minutes for removal of nuclei. Thesupernatant were separated by electrophoresis on 10% sodium dodecylsulfate-polyacrylamide gels (SDS-PAGE) under the reduced conditions. Theacrylamide concentration of the separation gels was 10% and appropriatemolecular weight markers were used. The electrophoretic transfer ofproteins to nitrocellulose membranes was done at 45 V for 16 hours.After protein transfer, the nitrocellulose membranes were incubated fortwo hours with blocking buffer containing 5% skim milk and 0.05%Tween-20 in PBS, and then incubated overnight with EB-1 antibody dilutedin blocking buffer at 1 μg/ml concentration. The membranes were rinsedthree times with wash buffer (PBS with 0.05% Tween 20) and incubated for1 h with affinity-purified goat anti-mouse immunoglobulin G conjugatedwith horseradish peroxidase diluted 1:3000 in blocking buffer. Afterthree washes, each reactive protein band was detected by enhancedchemiluminescence (ECL) kit manufactured by Amersham Pharmacia Biotech.FIG. 7 shows a SDA-PAGE and Western blotting analysis of thymocyte (A &B) and Molt-4 cell (C & D) lysate with EB-1 antibody. Lanes A and Crepresents the electrophoresis of cell lysate that is not treated withsialidase, whereas lanes B and D was the electrophoresis ofsialidase-treated lysate. EB-1 antibody recognized bothsialidase-treated and untreated CD43 molecules. This suggests that EB-1antibody might recognize unglycosylated region of CD43 molecule.

Example 9

In order to define the CD43 epitope recognized by the EB-1 antibody,CD43 mutants were constructed. The coding sequence for the human CD43gene was amplified from a CD43 cDNA and used for the construction ofexpression vectors. For example, to produce glutathione-S-transferase(GST)-fusion protein, CD43 recombinant plasmids were constructed bycloning PCR fragments into pGEX-2T vector manufactured by Pharmacia.Primers used for PCR amplification were selected based on the sequencein GeneBank and modified to contain BamHI and either EcoRI, or BglIIrestriction sites to facilitate cloning Both purified PCR products andpGEX-2T vector were digested with BamHI and either BglII or EcoRI at 37°C. overnight, ligated using T4 DNA ligase manufactured at 16° C.overnight, and then used to transform E. coli competent TOP1OF′ cells.FIG. 8 shows a schematic diagram of 11 CD43 deletion mutants. Forexample, the pGEX1-253 represents the pGEX-2T vector containing 1^(st)to 253^(rd) amino acids of human CD43.

To express GST-fusion proteins containing human CD43 sequences, E. coliTOP10 cells transformed with the recombinant plasmids were grown at 37°C. overnight in Luria troth (LB) medium containing 50 μg/ml ampicillin.The overnight cultured cells were diluted 20 times with fresh LB mediumcontaining the same concentration of ampicillin and grown at 37° C. for3 to 4 h until an optical density value of 0.6 was reached. The geneexpression was induced by adding IPTG into the culture to a finalconcentration of 1 mM. After 4 h of incubation at 37° C. with constantshaking, the cells were pelleted by centrifugation at 6000 g for 15minutes at 4° C. and then resuspended with 3 ml of lysis buffer (50 mMTris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl) for each gram of packed cells.The suspension was incubated on ice for 30 min with a finalconcentration of 0.2 mM PMSF.

For Western blotting of CD43 mutants with BB-1 antibody, aliquots ofeach lysate of GST-CD43 fusion protein were separated by electrophoresison 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels followed byblotting onto nitrocellulose membranes. The nitrocellulose membrane wasstained with EB-1 antibody or anti-GST polyclonal antibody and eachreactive protein band was detected by enhanced chemiluminescenceaccording to the procedure of Example 8. FIG. 9 shows the Westernblotting analysis of CD43 mutant with EB-1 (A) and anti-GST (B)antibodies. Lane 1 represent pGEX1-253, lane 2 pGEX1-98, lane 3pGEX1-87, lane 4 pGEX1-81, lane 5 pGEX1-75, lane 6 pGEX1-70, lane 7pGEX70-99, lane 8 pGEX71-81, lane 9 pGEX73-81, lane 10 pGEX76-81, lane11 pGEX73-80, lane 12 pGEX2T, and lane 13 human thymocyte lysate. Asshown in FIG. 9, pGEX73-81 contains the minimal sequence for recognitionof CD43 antigen by EB-1 and thus CD43 epitope for EB-1 antibody is73^(rd) to 81^(st) amino acid sequence of CD43. The sequence is asfollows, Glu Gly Ser Pro Leu Trp Thr Ser Ile (SEQ ID NO: 2). Therefore,this sequence is very useful in the aspect that the epitope recognizedby EB-1 antibody is not expressed on mature blood cell, hematopoieticstem cells, subsets of hematopoietic precursors in bone marrow or anynon-hematopoietic tissues.

Example 10

Example 3, 5, 6 and 7 show that EB-1 immunoreactivity is restricted tosubsets of hematopoietic precursors in bone marrow except forhematopoietic stem cells. In this example the expression of CD43 epitoperecognized by EB-1 antibody on leukemic cells were investigatedaccording to flow cytometric analysis of Example 3. Peripheral blood wascollected in EDTA tube from leukemia patients and erythrocytes andmature granulocyte were removed by centrifugation using Ficoll-Hypaquemanufactured Amersham Pharmacia Biotech. The purified cells were stainedwith FITC-conjugated EB-1 and analyzed using flow cytometer. Table 4below is the results of marker analysis of leukemic samples using EB-1antibody by flow cytometry. In 31 of 38 leukemia cases (81.6%), whichinclude acute myelogenous leukemia (AML), acute lymphogenous leukemia(ALL) and chronic myelogenous leukemia (CML) with blast crisis, tumorcells are stained by EB-1 antibody. FIG. 10 shows the representativeflow cytometric staining pattern of leukemic cells using EB-1 antibody.The solid line represents the staining by EB-1 antibody, and thenegative staining profile by an irrelevant antibody is shown as filledhistogram.

TABLE 4 EB-1 positivity No. of No. of Type of leukemia cases cases % AML22 20 90.9 M1 6 6 100.0 M2 8 7 87.5 M3 1 1 100.0 M4 4 4 100.0 Others 3 266.7 ALL 13 9 69.2 CML with blast crisis 3 2 66.7 Total 38 31 81.6

The present example was also carried out according toimmunohistochemical analysis of Example 2 to investigate whether EB-1antibody is reactive against lymphoblastic lymphoma cells using EB-1antibody purified from Example 4 as primary antibody. FIG. 11 shows therepresentative immunostaining pattern of lymphoblastic lymphoma tissueby EB-1 antibody. As a whole, 4 out of 9 (44.4%) lymphoblastic lymphomacases showed positive staining by EB-1 antibody.

Therefore, EB-1 antibody and its CD43 epitope could be powerfuldiagnostic and therapeutic tools for various types of acute leukemia,chronic leukemia with blast crisis and lymphoblastic lymphoma

Example 11

In order to determine the therapeutic potential of EB-1 antibody, EB-1immunotoxin and human leukemia model in mice were developed. To produceimmunotoxin, saporin manufactured by Sigma, and EB-1 antibody wereconjugated via a disulfide bond between chemically inserted sulfhydryl(SH) groups (Polito et al., 2004). The human leukemia model in mice wasestablished by the injection of 2×10⁵ CCRF-CEM cells in a 300 μl volumeof PBS into the tail vein of RAG-1-deficient mice. One week later, eachmouse was treated with four 100 μg doses of either of EB-1 antibody,EB-1-saporin immunotoxin, or irrelevant antibody as a bolus injection(in a 300 μl volume of PBS) into the tail vein in every other day (i.e.days 7, 9, 11 and 13). Four weeks after intravenous injection ofCCRF-CEM cells, blood sample from the retro-orbital plexus was stainedwith FITC-conjugated EB-1 antibody and PE-conjugated anti-human MHCclass I antibody according to flow cytometric analysis of Example 3 and10. FIG. 12 is representative result of flow cytometric analysis ofleukemic cells in blood from the mice. In control mice, 23.4% of cellsin peripheral blood was both human (CD43- and human MHC class I-positiveCCRF-CEM cells, where CCRF-CEM cells was reduced up to 1.3% in the mousetreated with EB-1-saporin immunotoxin. The leukemic cell burden was alsodecreased about two-fold in the mouse treated with EB-1 antibody only,compared with that in control mouse. Thus, EB-1 antibody could provideeffective tools for treatment of acute leukemic cells through thedelivery of toxic material into the tumor cells, antibody-dependent cellmediated cytotoxicity (ADCC), or other mechanisms.

Example 12

EB-1 antibody recognizes the 73^(rd) to 81^(st) amino acid sequence ofCD43, Glu Gly Ser Pro Leu Tip Thr Ser Ile (SEQ ID NO: 2). The presentexample was carried to develop new anti-CD43 antibodies that are usefulfor the diagnosis and treatment of leukemia and lymphoma. DNA fragmentencoding 70^(th) to 98^(th) amino acid sequence of CD43 that include SEQID NO:2 were cloned into pQE-40 vector, and then used to transformcompetent E. coli TOP10F′ cells. Transformed TOP10F′ cells were culturedaccording the methods of Example 9, and CD43 fusion protein was purifiedfrom the E. coli lysate by the passing of E. coli lysate through Nickelcolumn manufactured by Amersham Pharmacia Biotech. 100 μg of SY-CD43fusion protein was mixed with complete Freund's adjuvant andintraperitoneally administered and immunized into the Balb/c mice. Fourweeks later, additional two 100 μg doses of SY-CD43 fusion protein mixedwith incomplete Freund's adjuvant at two weeks intervalintraperitoneally administered. Three days after final boosting, thespleen was removed from the immunized mice and hybridoma cells wereprepared according the fusion method of Example 1. The culturesupernatant of hybridoma cells was screened using human CD43-transfectedmurine EL4 cells by flow cytometry, and two hybridoma clones that arereactive against CD43-transfected EL4 cells were selected and named asEB-2 and EB-3, respectively. FIG. 13 shows the flow cytometric analysisof CD43-transfected EL4 cell line, human thymocyte, human peripheralblood cells, and human acute leukemia cells by EB-2 and EB-3 antibodies.CD43-transfected EL4 cell line, human thymocytes and human leukemiccells were positive, but human peripheral blood cells were negative toboth EB-2 and EB-3 antibodies. Thus, staining pattern of both EB-2 andEB-3 antibodies is similar to that of EB-1.

Example 13

The present example was carried out to determine the CD43 epitoperecognized by EB-2 and EB-3 antibodies according the SDS-PAGE andWestern blotting of Example 9 using CD43 deletion mutants. FIG. 14 showsthe Western blotting analysis of CD43 mutant with EB-2 and EB-3. TheCD43 epitope for EB-3 antibodies is similar to that for EB 1. That is,the minimal epitope for both EB-1 and EB-3 antibodies are 73^(rd) to81^(st) amino acid sequence of CD43 (SEQ ID NO: 2; Glu Gly Ser Pro LeuTrp Thr Ser Ile). However, EB-2 antibody could recognize the smallerpeptide sequence than EB-1 and EB-3. The minimal epitope for EB-2 is76^(th) to 81⁵¹ amino acid sequence of CD43. The sequences is asfollows, Pro Leu Trp Thr Ser Ile (SEQ ID NO: 1).

The CD43 epitope of the present invention is useful for the diagnosis ofacute leukemia, chronic leukemia with blast crisis, and lymphoblasticlymphoma by determination whether this CD43 epitope is expressed upontissue examination or peripheral blood examination because this the CD43epitope of the present invention is not found in normalnon-hematopoietic tissue, mature blood cells, and activated peripheralblood except thymocytes. The CD43 epitope of the present invention canbe used as target material for the treatment of acute leukemia, chronicleukemia with blast crisis, and lymphoblastic lymphoma because it is notexpressed on hematopoietic stem cells and normal tissue exceptthymocytes and subsets of hematopoietic precursors in bone marrow.

The description of exemplary embodiments of biologically activepolypeptides is illustrative of the present invention. Because of thevariation which will be apparent to those skilled in the art, however,the present invention is not intended to be limited to the particularembodiments described above. The scope of the invention is defined inthe following claims.

1-30. (canceled)
 31. An isolated CD43 polypeptide less than 100 amino acids in length and including an amino acid sequence of SEQ ID NO:1.
 32. The isolated CD43 polypeptide of claim 31 that is less than 50 amino acids in length.
 33. The isolated CD43 polypeptide of claim 31 that includes an amino acid sequence of SEQ ID NO:2.
 34. The isolated CD43 polypeptide of claim 31, consisting of an amino acid sequence of SEQ ID NO:1.
 35. The isolated CD43 polypeptide of claim 31, consisting of an amino acid sequence of SEQ ID NO:2. 